The polypeptide hormone relaxin may be important in regulating spontaneous uterine contractile activity and cervical tone in many species, including human. The overall objective of this study is to delineate the moleculat mechanisms by which relaxin inhibits uterine activity. We have previously shown that while relaxin inhibited the spontaneous contractile activity of the estrogen-primed rat uterus in the absence of phosphodiesterase inhibitors, it elevated both protein-bound cAMP and total tissue cAMP levels, but only in the presence of methylisobutylxanthine. These facts, plus the finding that relaxation effects were more rapid in onset that were changes in cAMP levels (bound or total), suggested that the initial mechanisms by which relaxin suppresses spontaneous contractions probably does not involve cAMP. Alternatively, relaxin may bring about changes in intracellular calcium by a variety of mechanisms. In order to further probe the molecular aspects of relaxin action in the uterus, the effect of the hormone on calcium flux across the cell membrane, on microsomal calcium uptake, Ca++-ATPase, and specific phosphorylation, and on the phosphorylation/dephosphorylation ratios of contractile proteins such as myosin light chain and myosin light chain kinase will be studied. Uterine myosin, myosin light chains, and myosin light chain kinase will be purified and characterized with respect to the effect of phosphorylation on the activity of these proteins. Two-dimensional electrophoresis will be used to delineate the phosphorylation/dephosphorylation status of specific proteins after exposure of the tissue to relaxin. Parallel studies using other uterine relaxants with divergent actions (D600, isoproterenol, and dibutyryl cAMP) should allow further delineation of relaxation mechanisms in the uterus in general. Finally, the effect of relaxin on selected biochemical and morphological parameters in myometrial cells in culture will be examined.